The screening of the blood supply by the blood bank is expensive and labor intensive with the current state of the art being Gen-Probe's multiplex assay that has the potential to detect only two viral types simultaneously. Multiplex PCR offers a cost-effective solution, which, with refinement and full automation would allow screening of the donated blood supply, The major hurdle in the use of multiplex PCR as a detection protocol is the ability to accurately assign the identity of any one product in a potential mixture of many products, We propose to develop a single technology platform medical device with the potential to diagnose a broad variety of viral transfusion-transmitted disease organisms which may infect the human blood supply. This proposal leverages technology based on a high throughput, automated robotic system which performs the steps of cell lysis, nucleic acid extraction and purification, RT-PCR and detection of the PCR products by mass spectrometry. The exact mass of each PCR product in a mixture is used to determine the base composition of each product and this composition uniquely identifies all the organisms present in the original sample. This system utilizes a single-well multiplex PCR assay that will detect a large number of pathogens simultaneously. In phase one we propose to design and test primers for six blood banking pathogens (HIV types1 and 2, HTLV types 1 and 2, HBV and HCV). We will optimize the detection of each virus type singly in clinically relevant specimens (blood, serum and plasma) using single and multiplexed primer pairs. We will finally optimize the simultaneous detection of multiple pathogens in a single tube assay using multiplexed primer pairs.